The present invention relates to transporters, proteins involved in transport of substances from the outside to the inside of cells or vice versa.
Recently, the involvement of various transporters localized on the plasma membrane in the uptake system for nutrients and endogenous substances into cells and their transport mechanisms have been clarified (Tsuji, A. and Tamai, I., Pharm. Res., 13, 963-977, 1996). These transporters recognize the structures of substances to be transported to selectively transport specific substances. Transporters that recognize the relatively wide range of structures may possibly recognize foreign substances such as drugs by mistake, and actively take in them into cells. It is believed that drugs permeate through the plasma membrane fundamentally by simple diffusion depending on their physicochemical properties such as molecular size, hydrophobicity, and hydrogen-binding capacity. Particularly, in the case of ionic drugs, only molecules in the non-dissociated form can permeate through the plasma membrane according to the pH partition hypothesis. However, it has become evident that a number of drugs penetrate through the cell membrane by a specific mechanism other than simple diffusion, that is, an active transport mediated by transporters, in organs that require efficient exchange of intracellular and extracellular substances, including small intestine, uriniferous tubule, placenta, epithelial cells of choroid plexus, hepatocytes, and blood-brain barrier (Tamai, I. and Tsuji, A., Pharmacia, 31, 493-497, 1995; Saito, H. and Inui, K., Igaku no Ayumi, 179, 393-397, 1996; Tamai, I., Yakubutsu Dotai (Pharmacokinetics), 11, 642-650, 1996). For example, it is known that although oral xcex2-lactam antibiotics of the non-esterified type are amphoteric or negatively charged in physiological pHs and sparingly soluble in fat, they are readily absorbed through the intestine. The transport study using the isolated membrane-vesicles system demonstrated that an H+-driven peptide transporter localized on the brush-border is involved in the absorption process of these drugs (Tsuji, A. et al., J. Pharmacol. Exp. Ther., 241, 594-601, 1987). Although the specificity of a peptide transport system in terms of the peptide size is so strict as to recognize di- or tri-peptides but not tetrapeptides or larger peptides, it has a rather broad substrate specificity to recognize peptides comprising non-natural amino acids. The peptide transporter mediates transport of xcex2-lactam antibiotics mistakenly due to its broad substrate specificity. This property has been unexpectedly utilized in the clinical field (Tsuji, A., American Chemical Society (eds. Taylor, M. D., Amidon, G. L.), Washington, D.C., 101-134, 1995). Furthermore, a possibility that a transporter is also involved in permeation of substances with a high hydrophobicity such as fatty acids through the plasma membrane has been reported (Schaffer, J. and Lodish, H., Cell, 79, 427-436, 1994).
Since various transporters are supposed to be distributed in organs and cells based on the physiological roles of the organs and cells, their distribution and functions may be specific to organs. Therefore, transporters are expected to be used to impart an organ specificity to pharmacokinetics. In other words, an organ-specific drug delivery system (DDS) can be constructed utilizing transporters. If drug absorption solely relied on simple diffusion is improved by elevating its hydrophobicity, an effect of the drug obtained in the initial transport in the liver can be enhanced and the drug can non-specifically translocates into any organ. In addition, it would also be possible to increase the drug absorption independently of its fat-solubility by designing the drug utilizing the substrate specificity of transporters (Hayashi, K. et al., Drug Delivery System, 11, 205-213, 1996). For this purpose, it is necessary to identify various transporters at the molecular level and analyze their properties in detail. However, their molecular level identification are greatly behind studies on their membrane physiology because they are difficult to handle biochemically and require complicated processes in their functional assays.
Recently, cDNAs of several transporters have been cloned by the expression cloning method using Xenopus oocytes, a foreign gene expression system, and structural homology among them has been revealed (Fei, Y.-J. et al., Nature, 368, 563-566, 1994). For example, Koepsell et al. cloned an organic cation transporter, OCT1, which is assumed to be localized on a basement membrane, using the expression cloning method in 1994 (Grundemann, D. et al., Nature, 372, 549-552, 1994). Subsequently, OCT2 was identified by homology cloning based on the sequence of OCT1 (Okuda, M. et al., Biochem. Biophys. Res. Commun., 224, 500-507, 1996). OCT1 and OCT2 show homology as high as 67% to each other (Grundemann, D. et al., J. Biol. Chem., 272, 10408-10413, 1997). Both of them are intensely expressed in the kidney, but differ in the organ distribution; OCT1 is also expressed in the liver, colon, and small intestine, while OCT2 expression is specific to the kidney.
Only a few reports on identification of transporters at the molecular level, including the reports, are available, and there would be many unknown transporters that may be clinically useful.
An object of this invention is to provide a family of novel transporter genes, proteins encoded by these genes, and their use.
The present inventors have screened a fetal gene library constructed using the subtractive method by random sequencing based on a working hypothesis that fetal genes include those which are involved in various disorders including cancer and are specifically or intensely expressed in fetal tissues. The inventors discovered an unknown gene showing a significant homology with those for organic cation transporters, OCT1 and OCT2, and attempted to isolate this gene, which was assumed to encode a novel transporter. Thus, the inventors succeeded in isolating the desired gene by screening a cDNA library derived from human fetus. Furthermore, the inventors studied the transporter activity of a protein encoded by the isolated human gene and found that the protein, in fact, functioned as a transporter for various organic cations. The inventors also succeeded in isolating a mouse gene corresponding to the isolated human gene.
This invention relates to a family of novel transporter genes, proteins encoded by these genes, and their use, and more specifically to:
(1) a protein comprising an amino acid sequence set forth in SEQ ID NOs: 1, 3, 22, or 27, or a protein comprising said amino acid sequence in which one or more amino acid residues are substituted, deleted, or added, and having an activity to transport an organic cation;
(2) a protein encoded by a DNA hybridizing to a DNA comprising nucleotide sequence according to SEQ ID NOs: 2, 4, 23, or 28, and having an activity to transport an organic cation;
(3) a DNA encoding the protein according to (1) or (2);
(4) a vector comprising the DNA according to (3);
(5) a transformant expressibly carrying the DNA according to (3);
(6) a method for producing the protein according to (1) or (2), the method comprising culturing the transformant according to (5);
(7) an antibody that binds to the protein according to (1) or (2); and
(8) a DNA specifically hybridizing to a DNA comprising a nucleotide sequence set forth in SEQ ID NOs: 2, 4, 23, or 28, and consisting of at least 15 nucleotides.
Nucleotide sequences of cDNAs of novel human transporters isolated by the present inventors are shown in SEQ ID NO: 2 (designated as xe2x80x9chuman OCTN1xe2x80x9d) and SEQ ID NO: 4 (designated as xe2x80x9chuman OCTN2xe2x80x9d), respectively. Amino acid sequences of proteins encoded by these cDNAs are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. Amino acid sequences of these two proteins included in the transporter proteins of this invention showed such a high overall homology as about 76%, and both of them retained the following consensus sequence which is conserved in various types of transporters including the glucose transporter: [Leu, Ile, Val, Met, Ser, Thr, Ala, Gly]-[Leu, Ile, Val, Met, Phe, Ser, Ala, Gly]-Xaa less than 2 greater than -[Leu, Ile, Val, Met, Ser, Ala]-[Asp, Glu]-Xaa-[Leu, Ile, Val, Met, Phe, Tyr, Trp, Ala]-Gly-Arg-[Arg, Lys]-Xaa less than 4-6 greater than -[Gly, Ser, Thr, Ala] (Maiden, M. C. et al., Nature, 325, 641-643, 1987). In fact, these proteins have an activity to transport various organic cations (see Examples 6 to 8).
The present inventors also isolated mouse genes corresponding to the above-described human OCTN1 and human OCTN2. Nucleotide sequences of the isolated cDNAs are shown in SEQ ID NO: 23 (designated as xe2x80x9cmouse OCTN1xe2x80x9d) and SEQ ID NO: 28 (designated as xe2x80x9cmouse OCTN2xe2x80x9d), respectively. Amino acid sequences of proteins encoded by these cDNAs are shown in SEQ ID NOs: 22 and 27, respectively.
Transporter proteins of this invention also include those having the additional activity to transport substances other than organic cations as far as they retain the organic cation transport activity. Organic cations include, for example, TEA, carnitine, quinidine, and pyrilamine, but are not limited to them. They also include carcinostatic agents such as actinomycin D, etoposide, vinblastine, daunomycin, etc. Transporter proteins of this invention include those having the activity to transport organic cations not only from the outside to the inside of cells but also from the inside to the outside of cells.
Transporter proteins of this invention can be prepared as recombinant proteins using recombination techniques or natural proteins. Recombinant proteins can be prepared, for example, as described below, by culturing cells transformed with DNA encoding proteins of this invention. Natural proteins can be isolated from the kidney and cancer cell strains such as Hela S3, which highly express the proteins of this invention, by the method well known to those skilled in the art, for example, affinity chromatography using an antibody of this invention described below. The antibody may be either polyclonal or monoclonal. A polyclonal antibody can be prepared by purifying serum obtained from, for example, a small animal such as a rabbit immunized with proteins of this invention by known methods, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE-ion exchange column chromatography, affinity column chromatography coupled with the protein of this invention, etc. A monoclonal antibody can be prepared by immunizing a small animal such as a mouse with the protein of this invention, excising the spleen from the mouse, grinding the tissue into cells, fusing them with mouse myeloma cells using a fusing agent such as polyethylene glycol, and selecting a clone that produces an antibody to proteins of this invention out of fused cells (hybridomas) thus produced. Then, hybridomas thus selected are transplanted into the abdominal cavity of a mouse, and the ascites is collected from the mouse. A monoclonal antibody thus obtained can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE-ion exchange column chromatography, affinity column chromatography coupled with the protein of this invention, etc. When the antibody thus obtained is administered to human subjects, a humanized antibody or a human antibody is advantageously used to reduce the immunogenicity. An antibody can be humanized by, for example, the CDR grafting method comprising cloning an antibody gene from monoclonal antibody-producing cells and grafting the epitope portion thereof into an existing human antibody. A human antibody can be prepared by the usual method for preparing a monoclonal antibody except for immunizing a mouse whose immune system is replaced with the human""s.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which an amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
It is also possible for those skilled in the art to prepare proteins having functions equivalent to the transporter proteins of this invention (human OCTN1, human OCTN2, mouse OCTN1, and mouse OCTN2) by appropriately modifying amino acid residues of the proteins by, for example, substitution, using well known methods. Mutation of amino acids of the proteins can occur also spontaneously. Such mutant proteins which are obtained by altering the amino acid sequence of the transporter proteins of this invention by substitution, deletion, or addition of amino acid residues, and are functionally equivalent to those of the transporter proteins are also included in the proteins of this invention. Herein, xe2x80x9cfunctionally equivalentxe2x80x9d means that proteins have an activity to transport organic cations. Methods well known to those skilled in the art for altering amino acids include, for example, the site-specific mutagenesis system by PCR (GIBCO-BRL, Gaithersburg, Md.), site-specific mutagenesis by oligonucleotide (Kramer, W. and Fritz, H. J. (1987) Methods in Enzymol., 154: 350-367), Kunkel""s method (Methods Enzymol., 85, 2763-2766 (1988)), etc. The number of amino acids that can be substituted is usually 10 amino acid residues or less, preferably 6 or less, and more preferably 3 or less. The site of substitution, deletion, or addition of amino acid residues is not particularly limited as far as the activity of proteins of this invention is retained. It is possible to detect the transporter activity of proteins, for example, by the method described below in Example 6.
It is routine for those skilled in the art to obtain proteins functionally equivalent to the transporter proteins of this invention by isolating and using DNAs highly homologous to the DNA sequences encoding the transporter proteins of this invention (human OCTN1, human OCTN2, mouse OCTN1, and mouse OCTN2) or portions thereof using hybridization techniques (Sambrook, J. et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, 1989), etc. These proteins functionally equivalent to those of transporter proteins of this invention are also included in proteins of this invention. Here, xe2x80x9cfunctionally equivalentxe2x80x9d means that proteins have an activity to transport organic cations. DNAs that hybridize to the DNAs encoding the proteins of this invention can be isolated from other organisms, for example, rats, rabbits, cattle, etc. as well as humans and mice. Especially, tissues such as the kidney are suitable as sources of such DNAs. These DNAs isolated using hybridization techniques usually have a high homology with the above-described DNAs encoding the transporter proteins of this invention. xe2x80x9cHigh homologyxe2x80x9d means at least 70% or more, preferably at least 80% or more, and more preferably at least 90% or more of amino acid sequence identity. The xe2x80x9cpercent identityxe2x80x9d of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. Where gaps exist between two sequences, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.
One example of hybridization conditions for isolating such DNAs is as follows. That is, after the pre-hybridization at 55xc2x0 C. for 30 min or more in the xe2x80x9cExpressHyb Hybridization Solutionxe2x80x9d (CLONTECH), a labeled probe is added, and hybridization is performed by heating the reaction mixture at 3xc2x0 C. to 55xc2x0 C. for 1 h or more. Then, the reaction product is successively washed in 2xc3x97SSC and 0.1% SDS three times at room temperature for 20 min, and then in 1xc3x97SSC and 0.1% SDS once at 37xc2x0 C. for 20 min. More preferable conditions are as follows. After the pre-hybridization at 60xc2x0 C. for 30 min or more in the xe2x80x9cExpressHyb Hybridization Solutionxe2x80x9d (CLONTECH), a labeled probe is added, and hybridization is performed by heating the reaction mixture at 60xc2x0 C. for 1 h or more. Then, the reaction product is successively washed in 2xc3x97SSC and 0.1% SDS three times at room temperature for 20 min, and then in 1xc3x97SSC and 0.1% SDS twice at 50xc2x0 C. for 20 min. Still more preferable conditions are as follows. After pre-hybridization at 68xc2x0 C. for 30 min or more in the xe2x80x9cExpressHyb Hybridization Solutionxe2x80x9d (CLONTECH), a labeled probe is added, and hybridization is performed by heating the reaction mixture at 68xc2x0 C. for 1 h or more. Then, the reaction product is successively washed in 2xc3x97SSC and 0.1% SDS three times at room temperature for 20 min, and then in 0.1xc3x97SSC and 0.1% SDS twice at 50xc2x0 C. for 20 min.
The present invention also relates to DNAs encoding the above-described transporter proteins of this invention. DNAs of this invention may be cDNA, genomic DNAs, and synthetic DNAs. The DNAs of the present invention can be used for producing proteins of this invention as recombinant proteins. That is, it is possible to prepare proteins of this invention as recombinant proteins by inserting DNAs encoding proteins of this invention (e.g. DNAs comprising the nucleotide sequences set forth in SEQ ID NOs: 2, 4, 23, and 28) into an appropriate expression vector, culturing transformants obtained by transfecting suitable cells with the vector, and purifying the proteins thus expressed. Cells to be used for producing recombinant proteins include, for example, mammalian cells such as COS cells, CHO cells, NIH3T3 cells, etc., insect cells such as Sf9 cells, yeast cells, E. coli, and so on. Vectors used for the intracellular expression of recombinant proteins vary depending on host cells, including, for example, pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids Res., 1990, 18(7), p5322), etc. for mammalian cells, xe2x80x9cBAC-to-BAC baculovirus expression systemxe2x80x9d (GIBCO BRL), etc. for insect cells, xe2x80x9cPichia Expression Kitxe2x80x9d (Invitrogen), etc. for yeast cells, pGEX-5X-1 (Pharmacia), xe2x80x9cQIAexpress systemxe2x80x9d (Qiagen), etc. for E. coli. Host cells can be transformed with vectors, for example, by the calcium phosphate method, the DEAE-dextran method, the method using cationic liposome DOTAP (Boehringer Mannheim), the electroporation method, the calcium chloride method, etc. Recombinant proteins can be purified from recombinants thus obtained using standard methods, for example, as described in xe2x80x9cThe Qiaexpressionist Handbook, Qiagen, Hilden, Germany.xe2x80x9d
The present invention also relates to DNAs consisting of at least 15 nucleotides that specifically hybridize to the DNAs encoding proteins of this invention. Herein, xe2x80x9cspecifically hybridizexe2x80x9d means that a DNA does not cross-hybridize to other DNAs encoding other proteins under usual hybridization conditions, preferably under the stringent hybridization conditions. Such a DNA can be utilized as a probe for detecting and isolating DNA encoding the protein of this invention, and as a primer for amplifying the DNA.
By hybridization under xe2x80x9cstringent conditionsxe2x80x9d is meant hybridization at 37xc2x0 C., 1xc3x97SSC, followed by washing at 42xc2x0 C., 0.5xc3x97SSC.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
The transporter proteins of this invention can be used to control internal absorption and dynamics of drugs. Based on the results of detailed analysis of the substrate specificity of transporter proteins of this invention, drugs can be designed so as to be transported by these transporters and absorbability of the drugs mediated by these transporter proteins can be improved. Conventional modifications to enhance hydrophobicity are no longer necessary for drugs so designed, which enables speedily and efficiently developing water-soluble drugs that are easy to handle. The drugs thus developed is thought to be absorbed principally depending on the internal distribution pattern of transporter proteins of this invention, and an organ-specific delivery of the drugs thus becomes possible. Especially, if the transporter proteins of this invention are distributed in the target organ of a drug, an ideal drug delivery system (DDS) can be developed. If a drug is to be absorbed mediated by not the transporter proteins of this invention but other transporters, the drug can be designed so as to be specific to other transporter proteins by designing it considering the substrate specificity of the transporter proteins of this invention. Since the transporter proteins of this invention are present in the kidney, it is possible to reduce the nephrotoxicity produced by a drug by designing the drug so that it can be readily excreted by the transporter proteins of this invention.
Another possible application of this invention is to develop a drug targeting the transporter proteins of this invention. The transporters play important roles in the absorption mechanism of nutrients and drugs, or the excretion mechanism of drugs and internal metabolites. Thus, damage or abnormal elevation of the transporter""s functions may cause some disorders. It is considered to be efficacious against such disorders to administer a drug containing a compound that inhibits or enhances functions of the transporter proteins of this invention, or regulates the expression level of the transporter gene of this invention and the amount of the transporter proteins. The DNAs of this invention can be used in gene therapy for disorders caused by abnormalities in the activity and expression of the proteins of this invention. In this case, the DNA of this invention are inserted to an adenovirus vector (e.g. pAdexLcw), a retrovirus vector (e.g. pZIPneo), etc., and administered into the body by either ex vivo method or in vivo method. Gene therapy can also be performed by administering a synthetic antisense DNA to the body either directly or after inserted into the above-described vector.
Especially, since xe2x80x9cOCTN2xe2x80x9d included in the transporter proteins of this invention efficiently transports carnitine, chemotherapy with compounds to control the activity of xe2x80x9cOCTN2xe2x80x9d or gene therapy using the xe2x80x9cOCTN2xe2x80x9d gene is considered to be efficacious against various pathological conditions such as fatty liver, myocardiopathy, myopathy, etc. caused by hypocarnitinemia.
The transporter proteins of this invention are expressed in a variety of cancer cell strains, which suggests that the proteins may transport drugs into tumor cells. If this is the case, it is possible to develop carcinostatics that will be readily absorbed mediated by the transporter proteins of this invention. On the contrary, mechanisms to transport and excrete substances by the transporter proteins of this invention may function to excrete carcinostatics in tumor cells so that the cells acquire resistance to drugs. If the transporter proteins of this invention are involved in a mechanism of tumor cells to acquire drug resistance, a carcinostatic effect can be enhanced by a combined use of inhibitors of the transporter proteins of this invention with carcinostatics.